Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis
The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back translating the cDNA; some of these may be relevan...
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Language: | English |
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PORTLAND PRESS
1996
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Online Access: | http://hdl.handle.net/11573/406686 http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=A1996TW99200011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=0c7ff228ccbaaa74236f48834a34396a http://www.scopus.com/inward/record.url?eid=2-s2.0-0030056291&partnerID=65&md5=2a4585f941ff361388f8046c958b6eb8 |
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ftunivromairis:oai:iris.uniroma1.it:11573/406686 2024-04-21T08:12:21+00:00 Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis CUTRUZZOLA', Francesca TRAVAGLINI ALLOCATELLI, Carlo BRUNORI, Maurizio A. Brancaccio Cutruzzola', Francesca TRAVAGLINI ALLOCATELLI, Carlo A., Brancaccio Brunori, Maurizio 1996 http://hdl.handle.net/11573/406686 http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=A1996TW99200011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=0c7ff228ccbaaa74236f48834a34396a http://www.scopus.com/inward/record.url?eid=2-s2.0-0030056291&partnerID=65&md5=2a4585f941ff361388f8046c958b6eb8 eng eng PORTLAND PRESS info:eu-repo/semantics/altIdentifier/pmid/8660313 info:eu-repo/semantics/altIdentifier/wos/WOS:A1996TW99200011 volume:314 issue:1 firstpage:83 lastpage:90 numberofpages:8 journal:BIOCHEMICAL JOURNAL http://hdl.handle.net/11573/406686 info:eu-repo/semantics/altIdentifier/scopus/2-s2.0-0030056291 http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=A1996TW99200011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=0c7ff228ccbaaa74236f48834a34396a http://www.scopus.com/inward/record.url?eid=2-s2.0-0030056291&partnerID=65&md5=2a4585f941ff361388f8046c958b6eb8 info:eu-repo/semantics/article 1996 ftunivromairis 2024-03-28T01:55:23Z The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back translating the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved. Article in Journal/Newspaper Sperm whale Sapienza Università di Roma: CINECA IRIS |
institution |
Open Polar |
collection |
Sapienza Università di Roma: CINECA IRIS |
op_collection_id |
ftunivromairis |
language |
English |
description |
The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back translating the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved. |
author2 |
Cutruzzola', Francesca TRAVAGLINI ALLOCATELLI, Carlo A., Brancaccio Brunori, Maurizio |
format |
Article in Journal/Newspaper |
author |
CUTRUZZOLA', Francesca TRAVAGLINI ALLOCATELLI, Carlo BRUNORI, Maurizio A. Brancaccio |
spellingShingle |
CUTRUZZOLA', Francesca TRAVAGLINI ALLOCATELLI, Carlo BRUNORI, Maurizio A. Brancaccio Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
author_facet |
CUTRUZZOLA', Francesca TRAVAGLINI ALLOCATELLI, Carlo BRUNORI, Maurizio A. Brancaccio |
author_sort |
CUTRUZZOLA', Francesca |
title |
Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
title_short |
Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
title_full |
Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
title_fullStr |
Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
title_full_unstemmed |
Aplysia limacina myoglobin cDNA cloning: An alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
title_sort |
aplysia limacina myoglobin cdna cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis |
publisher |
PORTLAND PRESS |
publishDate |
1996 |
url |
http://hdl.handle.net/11573/406686 http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=A1996TW99200011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=0c7ff228ccbaaa74236f48834a34396a http://www.scopus.com/inward/record.url?eid=2-s2.0-0030056291&partnerID=65&md5=2a4585f941ff361388f8046c958b6eb8 |
genre |
Sperm whale |
genre_facet |
Sperm whale |
op_relation |
info:eu-repo/semantics/altIdentifier/pmid/8660313 info:eu-repo/semantics/altIdentifier/wos/WOS:A1996TW99200011 volume:314 issue:1 firstpage:83 lastpage:90 numberofpages:8 journal:BIOCHEMICAL JOURNAL http://hdl.handle.net/11573/406686 info:eu-repo/semantics/altIdentifier/scopus/2-s2.0-0030056291 http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=A1996TW99200011&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=0c7ff228ccbaaa74236f48834a34396a http://www.scopus.com/inward/record.url?eid=2-s2.0-0030056291&partnerID=65&md5=2a4585f941ff361388f8046c958b6eb8 |
_version_ |
1796932376976162816 |